Aptamer-Functionalized Ce4+-Ion-Modified C-Dots: Peroxidase Mimicking Aptananozymes for the Oxidation of Dopamine and Cytotoxic Effects toward Cancer Cells

Aptamer-functionalized Ce4+-ion-modified C-dots act as catalytic hybrid systems, aptananozymes, catalyzing the H2O2 oxidation of dopamine. A series of aptananozymes functionalized with different configurations of the dopamine binding aptamer, DBA, are introduced. All aptananozymes reveal substantially enhanced catalytic activities as compared to the separated Ce4+-ion-modified C-dots and aptamer constituents, and structure–catalytic functions between the structure and binding modes of the aptamers linked to the C-dots are demonstrated. The enhanced catalytic functions of the aptananozymes are attributed to the aptamer-induced concentration of the reaction substrates in spatial proximity to the Ce4+-ion-modified C-dots catalytic sites. The oxidation processes driven by the Ce4+-ion-modified C-dots involve the formation of reactive oxygen species (•OH radicals). Accordingly, Ce4+-ion-modified C-dots with the AS1411 aptamer or MUC1 aptamer, recognizing specific biomarkers associated with cancer cells, are employed as targeted catalytic agents for chemodynamic treatment of cancer cells. Treatment of MDA-MB-231 breast cancer cells and MCF-10A epithelial breast cells, as control, with the AS1411 aptamer- or MUC1 aptamer-modified Ce4+-ion-modified C-dots reveals selective cytotoxicity toward the cancer cells. In vivo experiments reveal that the aptamer-functionalized nanoparticles inhibit MDA-MB-231 tumor growth.


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Instruments. Absorption spectra were recorded on a UV-2450 spectrophotometer (UV, Shimadzu). Kinetic measurements were performed at 25 °C using a Biotek Synergy H1 microplate reader, equipped with a Biotek dual dispensing unit, and using Corning 3696 96-hald-well plates. Dissociation constants were evaluated using Isothermal titration calorimetry (ITC) instrument (Malvern instruments MicroCal PEAQ-ITC). X-ray photoelectron spectroscopy (XPS) measurements were performed with an Axis Ultra photoelectron spectrometer from Kratos Analytical. Fourier-transform infrared spectroscopy (FTIR) measurements were performed using a Nicolet iS50 FTIR Spectrometer. Transmission Electron Microscope was performed on a Tecnai G2 Spirit TWIN T12. Ce 4+ -ions-modified C-dots were characterized by coupled plasma mass spectrometry using an Inductively Coupled Plasma-Mass Spectrometry (ICP) instrument. Electron paramagnitic resonance (EPR) measurements were carried out at room temperature using a Bruker ELEXYS E500 spectrometer operating at X-band frequencies (9.5 GHz) and a Bruker ER4102ST resonator.
Preparation of C-dots and their modification with Ce 4+ -ions. The C-dots were synthesized according to Wang et al 1 . Citric acid and urea were mixed in water and heated for 4-5 min in a 750 W microwave. This solid was then transferred to a vacuum oven and heated at 60℃ for 1 h to remove the residual small molecules. An aqueous solution of the C-dots was purified in a centrifuge (3000 g, 20 min) to remove large or agglomerated particles. The resulting colored (brown) aqueous solution remained indefinitely stable at various concentrations. The obtained C-dots solution was purified by 3 kDa and 10 kDa cutoff filters under 9168 g for 30 min, respectively. Finally, the S3 C-dots solution of below 3 kDa was frozen by liquid nitrogen and dried through lyophilization. The Ce 4+ -ion-modified C-dots were prepared as follows: ammonium cerium (IV) nitrate (0.2 M, 50 μL) and C-dots (10 mg mL −1 , 500 μL) were mixed and shaking for 30 min. The mixture was under centrifugation at 18000 g for 5 min to collect the sediment. The wash process is repeated for several times.

Modification of Ce 4+ -ion-modified C-dots with amino-modified aptamers or amino-functionalized control strands.
A 50 uL solution of Ce 4+ -ion-modified C-dots (4 mg mL −1 ) were subsequently added to equal volumes of 20 μL EDC (50 mM) and 50 μL sulfo-NHS (50 mM) in MES buffer (5 mM, pH 5.5) and left to react for 15 min. The above mixture was sequentially added 100 μL of 100 mM phosphate buffer, pH 7.2 and 100 μL of amino-functionalized aptamer (20 μM in 100 mM phosphate buffer, pH 7.2). The coupling reaction was performed at room temperature for 12 h. The solution after the reaction was purified by 3 kDa cutoff filters under 10000 g for 30 min and repeat for 4 times.
Evaluation of the loading of the aptamers on Ce 4+ -ion-modified C-dots. After loading the amino-functionalized aptamers to the Ce 4+ -ion-modified C-dots, the concentration of the Ce 4+ -ions was determined by the ICP. Following the same procedure of preparation of attaching the aptamers to Ce 4+ -ion-modified C-dots, the ratios of C-dots to Ce 4+ -ions are very similar to each other (1 : 0.3, mg mL −1 ).
The ratios between the Ce 4+ -ion-modified C-dots and aptamer could be determined by the relative calibration curves. As we can see from the Figure S4A and S4B, the two calibration curves at 260 and 338 nm were formed by the different concentrations of S4 the Ce 4+ -modified C-dots solution by UV spectra. Figure S4C show the UV spectra of 5-DBA associated with Ce 4+ -ion-modified C-dots. After we attach the NH 2 -aptamers to the C-dots, the calibration curve at 330 nm is required to quantify the amount of Cdots, while the absorbance of C-dots at 260 nm is obtained. By the subtraction of the spectrum at 260 nm, the net absorbance of the nucleic acids associated with the C-dots is evaluated. It should be noted that multiple groups of experiments were executed to achieve the similar ratio of aptamers to Ce 4+ -ion-modified C-dots. MDA-MB-231 of 6X106 cells/mouse were injected subcutaneously to the flack of each mouse. Tumor mass was generated after 7 days in a volume that is around 80-100 mm 3 , then the injections of the treatment were done intra-tumoral (IT) (2 times/week) in total 7 injections, by using C-dot-Ce 4+ for the control group, compared to MUC-1 modified S8 C-dot-Ce 4+ or AS1411-modified C-dot-Ce 4+ , all particles were injected in a volume of 100 µL of the amount of 50µg/mouse. NPs were prepared in a final concentration of 1 mg mL -1 . For each group we used 4 mice. Tumor was majored every 3 days before the following injection to evaluate the width and the height, then tumor volume (mm3) was measured using the equation of (Width2XHigeht)/2. Toxicity of the treatment was evaluated by the mice weight change (g) that was measured once a week. All results were presented as mean ± SEM.  Figure S2. FTIR spectra of (i) C-dots, and (ii) Ce 4+ -ions-modified C-dots.